Nicotinamide adenine dinucleotide (NAD) and its derivative compounds are known as essential coenzymes in cellular redox reactions in all living organisms. Several lines of evidence have also shown that NAD participates in a number of important signaling pathways in mammalian cells, including poly(ADP-ribosyl)ation in DNA repair (Menissier de Murcia et al., EMBO J., (2003) 22, 2255-2263), mono-ADP-ribosylation in the immune response and G protein-coupled signaling (Corda and Di Girolamo, EMBO J., (2003) 22, 1953-8), and the synthesis of cyclic ADP-ribose and nicotinate adenine dinucleotide phosphate (NAADP) in intracellular calcium signaling (Lee, Annu. Rev. Pharmacol. Toxicol., (2001) 41, 317-345). Recently, it has also been shown that NAD and its derivatives play an important role in transcriptional regulation (Lin and Guarente, Curr. Opin. Cell. Biol., (2003) 15, 241-246). In particular, the discovery of Sir2 NAD-dependent deacetylase activity (e.g., Imai et al., Nature, (2000) 403, 795-800; Landry et al., Biochem. Biophys. Res. Commun., (2000) 278, 685-690; Smith et al., Proc. Natl. Acad. Sci. USA, (2000) 97, 6658-6663) drew attention to this new role of NAD.
The Sir2 family of proteins consumes NAD for its deacetylase activity and regulates transcription by deacetylating histones and a number of other transcription regulators (see FIG. 1). Because of this absolute requirement for NAD, it has been proposed that Sir2 proteins function as energy sensors that convert the energy status of cells to the transcriptional regulatory status of genes (Imai et al., Nature, (2000) 403, 795-800; Imai et al., Cold Spring Harbor Symp. Quant. Biol., (2000) 65, 297-302). Sir2 proteins produce nicotinamide and O-acetyl-ADP-ribose in addition to the deacetylated protein substrates in their deacetylation reaction (Moazed, Curr. Opin. Cell. Biol., (2001)13, 232-238; Denu, Trends Biochem. Sci., (2003) 28, 41-48; see also FIG. 1), and nicotinamide is eventually recycled into NAD biosynthesis. Unlike other NAD-dependent biochemical reactions, the NAD-dependent deacetylase activity of the Sir2 family of proteins is generally highly conserved from bacteria to mammals (Frye, Biochem. Biophys. Res. Commun., (2000) 273, 793-798), suggesting that the connection between NAD and Sir2 proteins is ancient and fundamental. In mammals, the Sir2 ortholog, Sirt1/Sir2α, has been shown to regulate metabolism in response to nutrient availability (Bordone and Guarente, Nat. Rev. Mol. Cell Biol., (2005) 6, 298-305). In adipocytes, Sirt1 triggers lipolysis and promotes free fatty acid mobilization by repressing PPAR-γ, a nuclear receptor that promotes adipogenesis (Picard et al., Nature, (2004) 429, 771-776). In hepatocytes, Sirt1 regulates the gluconeogenic and glycolytic pathways in response to fasting by interacting with and deacetylating PGC-1α, a key transcriptional regulator of glucose production in the liver (Rodgers et al., Nature, (2005) 434, 113-118). Additionally, Sirt1 promotes insulin secretion in pancreatic β cells in response to high glucose partly by repressing Ucp2 expression and increasing cellular ATP levels (Moynihan et al., Cell Metab., (2005) 2, 105-117). While little is known about the regulation of NAD biosynthesis in mammals, NAD biosynthesis may play a role in the regulation of metabolic responses by altering the activity of certain NAD-dependent enzymes such as Sirt1 in a variety of organs and/or tissues.
The NAD biosynthesis pathways have been characterized in prokaryotes by using Escherichia coli and Salmonella typhimurium (Penfound and Foster, Biosynthesis and recycling of NAD, in Escherichia coli and Salmonella: Cellular and Molecular Biology, p. 721-730, ed. Neidhardt, F. C., 1996, ASM Press: Washington, D.C.) and recently in yeast (Lin and Guarente, Curr. Opin. Cell. Biol., (2003) 15, 241-246; Denu, Trends Biochem. Sci., (2003) 28, 41-48). In prokaryotes and lower eukaryotes, NAD is synthesized by the de novo pathway via quinolinic acid and by the salvage pathway via nicotinic acid (see FIG. 2) (Penfound and Foster, id.) In yeast, the de novo pathway begins with tryptophan, which is converted to nicotinic acid mononucleotide (NaMN) through six enzymatic steps and one non-enzymatic reaction (Lin and Guarente, Curr. Opin. Cell. Biol., (2003) 15, 241-246). Two genes, BNA1 and QPT1, have been characterized in this pathway in yeast. At the step of NaMN synthesis, the de novo pathway converges with the salvage pathway (see FIG. 2). The salvage pathway begins with the breakdown of NAD into nicotinamide and O-acetyl-ADP-ribose, which is mainly catalyzed by the Sir2 proteins in yeast. Nicotinamide is then deamidated to nicotinic acid by a nicotinamidase encoded by the PNC1 gene. Nicotinic acid phosphoribosyltransferase (Npt), encoded by the NPT1 gene, converts nicotinic acid to NaMN, which is eventually converted to NAD through the sequential reactions of nicotinamide/nicotinic acid mononucleotide adenylyltransferase (encoded by NMA1 and/or NMA2) and NAD synthetase (encoded by QNS1).
It has been shown that the NAD salvage pathway plays an important role in regulating Sir2 activity in yeast (Lin et al., Nature, (2002) 418, 344-348; Anderson et al., J. Biol. Chem., (2002) 277, 18881-18890; Anderson et al., Nature, (2003) 423, 181-185). For example, increased dosage of NPT1 increases Sir2-dependent transcriptional silencing and extends the life span of yeast mother cells (Anderson et al., J. Biol. Chem., (2002) 277, 18881-18890). Consistent with this finding, deletion of NPT1 causes a loss of Sir2-dependent silencing (Sandmeier et al., Genetics, (2002) 160, 877-889). Additional copies of other salvage pathway genes, PNC1, NMA1, and NMA2, have also been shown to increase telomeric and rDNA silencing (Anderson et al., J. Biol. Chem., (2002) 277, 18881-18890), while deletions of the de novo pathway genes, BNA1 or QPT1, have also been shown to have no effect on silencing at these loci (Sandmeier et al., Genetics, (2002) 160, 877-889). It has also been shown that PNC1 may be induced by different types of stress, including caloric restriction, and plays a critical role in regulating Sir2 activity in yeast (Anderson et al., Nature, (2003) 423, 181-185). These findings suggest that the regulation of NAD biosynthesis may play a role in Sir2-mediated transcriptional silencing and longevity control in yeast.
In vertebrates, NAD biosynthesis is markedly different from that of yeast and invertebrates (see FIG. 3). It is known that mammals predominantly use nicotinamide rather than nicotinic acid as a precursor for NAD biosynthesis (Magni et al., Adv. Enzymol. Relat. Areas Mol. Biol., (1999) 73, 135-182). Despite significant numbers of studies about NAD biosynthesis in the 1950's and 1960's, mammalian NAD biosynthesis enzymes have been generally poorly characterized. For example, it was not until 2001 that human nicotinamide/nicotinic acid mononucleotide adenylyltransferase (Nmnat), an enzyme required to convert NMN and NaMN to NAD in the nucleus (Hogeboom et al., J. Biol. Chem., (1952) 197, 611-620), was finally isolated and fully characterized (Emanuelli et al., J. Biol. Chem., (2001) 276, 406-412; Schweigler et al., FEBS Lett., (2001) 492, 95-100). Other critical enzymes in mammalian NAD biosynthesis pathways have yet to be characterized, thus the regulation of NAD biosynthesis is still relatively poorly understood in mammals.
Nampt has very ancient origins as an NAD biosynthesis enzyme. The entire pyridine nucleotide salvage cycle containing Nampt, Nmnat, and Sir2 homologues has been shown to exist even in the vibriophage (Miller et al., J. Bacteriol., (2003) 185, 5220-5233). Despite its ancient origins, Nampt has a relatively peculiar phylogenetic distribution. No other organisms between bacteria and vertebrates have obvious homologs of Nampt, except for one sponge species, and the homology of Nampt proteins between bacteria and vertebrates is unusually high (Revollo et al., J. Biol. Chem., (2004) 279, 50754-50763). Interestingly, the organisms that do not have Nampt homologs, such as yeast, worms, and flies, typically have nicotinamidase (Pnc1) homologs (Ghislain et al., Yeast, (2002) 19, 215-324.). It is likely that the organisms that have nicotinamidase use nicotinic acid as a precursor for NAD biosynthesis, while the organisms that have Nampt use nicotinamide as the main precursor for NAD biosynthesis. Because no obvious homologues of Pnc1 have been found in vertebrates (Rongvaux et al., Bioessays, (2003) 25, 683-690), the presence of Nampt, which allows a more direct pathway for NAD biosynthesis from nicotinamide (see FIG. 2), distinguishes the NAD biosynthesis in vertebrates from that in yeast and invertebrates.
The gene encoding human Nampt was originally isolated as a presumptive cytokine named pre-B cell colony-enhancing factor (PBEF) (Samal et al., Mol. Cell. Biol., (1994) 14, 1431-1437), although the PBEF function has never been reproduced. Since then, other groups have also shown that PBEF is indeed mammalian Nampt (Revollo et al., J. Biol. Chem., (2004) 279, 50754-50763; Rongvaux et al., Eur. J. Immunol., (2002) 32, 3225-3234; van der Veer et al., Circ. Res., (2005) 97, 25-34). Recently, Nampt/PBEF has been re-identified as a “new visceral fat-derived hormone” named visfatin (Fukuhara et al., Science, (2005) 307, 426-430). Fukuhara et al. report that visfatin is enriched in the visceral fat of both humans and mice and that its plasma levels increase during the development of obesity. Fukuhara et al. report that visfatin exerts insulin-mimetic effects in cultured cells and lowers plasma glucose levels in mice by binding to and activating the insulin receptor. However, the physiological relevance of visfatin is still in question because its plasma concentration is 40 to 100-fold lower than that of insulin. Additionally, Fukuhara et al. did not describe any connections between visfatin and Nampt. In Nampt/visfatin-deficient heterozygous mice, impaired glucose tolerance was observed, and Fukuhara et al. described that this phenotype is due to the insufficient insulin-mimetic function of visfatin. Alternatively, however, it is possible that the phenotype is actually due to insufficient NAD biosynthesis in the heterozygous mice, resulting in relatively insufficient activity of critical NAD-dependent enzymes involved in the regulation of glucose metabolism, such as Sirt1. Fukuhara et al. did not examine this possibility, nor did they report insulin levels of the mice during intraperitoneal glucose tests. Additionally, it has recently been reported that certain common polymorphisms in the promoter of the Nampt/PBEF/visfatin gene are associated with fasting insulin levels in a perfect linkage disequilibrium, but not with type 2 diabetes, in a French-Canadian population (Bailey et al., Diabetes, (2006) 55, 2896-2902.
Although a number of papers have been published since this first report of visfatin, the results are contradictory; the physiological relevance of visfatin, therefore, is still in question (Sethi et al., Trends. Mol. Med., (2005) 11, 344-347; Arner, J. Clin. Endocrinol. Metab., (2006) 91, 28-30; Stephens et al., Curr. Opin. Lipidol., (2006) 17, 128-131). For example, one study reported that plasma visfatin concentrations correlate with BMI and percent body fat but not with visceral fat mass or waist-to-hip ratio (Berndt et al., Diabetes, (2005) 54, 2911-2916). Another study reported that plasma visfatin is reduced in human obesity and is not related to insulin resistance (Pagano et al., J. Clin. Endocrinol. Metab., (2006) 91, 3165-3170). On the other hand, still another study reported that increasing plasma visfatin levels are independently and significantly associated with type 2 diabetes even after adjusting known biomarkers (Chen et al., J. Clin. Endocrinol. Metab., (2006) 91, 295-299). Accordingly, it is important to understand whether the NAD biosynthesis function or the insulin-mimetic function is more physiologically relevant in the regulation of glucose metabolism in mammals. It would also be desirable to provide processes and materials useful in NAD biosynthesis and/or the regulation of glucose metabolism in mammals.